The muscle of golden freshwater clam was extracted using hot water. The residual meat was freeze-dried then hydrolyzed at 50 degrees C first by Protamex (PX) as primary hydrolysis followed by a secondary hydrolysis (Flavourzyme, F). The inhibitory effects of hot water extracts and hydrolysates against angiotensin I converting enzyme (ACE) were investigated. The soluble protein content and peptide content of the hot water extracts was 85 and 84 mg/g, respectively. The IC50 on ACE was 1.95 mg/ml. The highest values of soluble protein (571 mg/g) and peptide content (272 mg/g) of hydrolysate prepared by PX hydrolysis for 5 h (PX5) or PX hydrolysis for 5 h followed by F hydrolysis for 0.5 h (PX5F0.5). PX5 showed the lowest IC50 on ACE (0.043 mg/ml), and a mixed-type inhibition kinetics while Captopril showed competitive inhibition. Their K-i values were 0.032 mg/ml and 0.0067 mu g/ml, respectively. The PX5 hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25. A fraction of the PX5 had molecular weight ranged 420-380 Da showed the highest inhibitory efficiency ratio (IER) being 1314.7%/mg/ml. The amino acid sequences of the two peaks of this fraction PX-D1 and PX-D2 were Val-Lys-Pro and Val-Lys-Lys, of which IC50 value were 3.7 and 1045 mu M, respectively. This freshwater clam hydrolysate (PX5) (peptide concentration 5 mg/ml) was used as drinks administered to spontaneously hypertensive rats (SHR) for 8 weeks. The systolic blood pressure and diastolic blood pressure of the SHR were significantly reduced by 22.0 and 13.2 mmHg, respectively, indicating a hypotensive effect by oral administration. (c) 2006 Elsevier Ltd. All rights reserved.